Characterization of Salmonella Type III Secretion Hyper-Activity Which Results in Biofilm-Like Cell Aggregation

We have previously reported the cloning of the Salmonella enterica serovar Typhimurium SPI-1 secretion system and the use of this clone to functionally complement a ΔSPI-1 strain for type III secretion activity. In the current study, we discovered that S. Typhimurium cultures containing cloned SPI-1 display an adherent biofilm and cell clumps in the media. This phenotype was associated with hyper-expression of SPI-1 type III secretion functions. The biofilm and cell clumps were associated with copious amounts of secreted SPI-1 protein substrates SipA, SipB, SipC, SopB, SopE, and SptP. We used a C-terminally FLAG-tagged SipA protein to further demonstrate SPI-1 substrate association with the cell aggregates using fluorescence microscopy and immunogold electron microscopy. Different S. Typhimurium backgrounds and both flagellated and nonflagellated strains displayed the biofilm phenotype. Mutations in genes essential for known bacterial biofilm pathways (bcsA, csgBA, bapA) did not affect the biofilms formed here indicating that this phenomenon is independent of established biofilm mechanisms. The SPI-1-mediated biofilm was able to massively recruit heterologous non-biofilm forming bacteria into the adherent cell community. The results indicate a bacterial aggregation phenotype mediated by elevated SPI-1 type III secretion activity with applications for engineered biofilm formation, protein purification strategies, and antigen display

Recruitment of non-biofilm cells into SPI-1 biofilm.

<p><b><u>Panel A</u>:</b> Non-biofilm strain χ3339 <i>flhCD</i> expressing GFP and strain χ3339 <i>flhCD</i> (R995 + SPI-1) were co-cultured, and the resulting biofilm was visualized using fluorescence microscopy. Controls consisted of each strain cultured separately and biofilm post-treatment with the GFP strain as described in the text. Biofilm post-treatment consisted of exposing the surface of the pre-formed, adhered biofilm with the non-biofilm GFP strain for approximately 10 minutes followed by washing of the sample to remove non-adhered cells. This treatment is not sufficient for incorporation of the GFP strain into the organized biofilm and indicates that simultaneous growth of the two strains is required for this to occur. <b><u>Panel B</u></b>: The same experiment as in Panel A was performed, except non-biofilm <i>E. coli</i> strain ATCC25922 expressing GFP was cultured with strain χ3339 <i>flhCD</i> (R995 + SPI-1). Controls not shown gave the same results as in Panel A.</p

SDS-PAGE of SPI-1 biofilm.

<p>The following samples from χ3339 <i>flhCD</i> (R995 + SPI-1) culture were run in an SDS-PAGE gel and Coomassie stained: <b><u>Lane A</u></b>: total cell lysate (from approximately 0.5 ml of culture); <b><u>Lane B</u></b>: culture supernatant (from approximately 0.1 ml of culture); <b><u>Lanes C and D</u></b>: biofilms harvested from replicate samples corresponding to 2 independent cultures of the R995 + SPI-1 strain. The indicated bands were excised and identified via mass spectrometry (MS) analysis. The identification of each protein band and its corresponding molecular weight (MW) are provided.</p

TEM analysis of samples from χ3339 <i>flhCD</i> (R995) culture and χ3339 <i>flhCD</i> (R995 + SPI-1) biofilm.

<p><b><u>Panels A and B:</u></b> Cells from R995 culture were harvested, washed in PBS, and visualized for TEM as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033080#s4" target="_blank">Materials and Methods</a>. <b><u>Panels C and D:</u></b> Cells from R995 + SPI-1 biofilm were harvested and visualized for TEM as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033080#s4" target="_blank">Materials and Methods</a>. Note the presence of a “sheath” material on the surface of the R995 + SPI-1 biofilm cells. All size bars = 500 nm.</p

SPI-1 biofilm in flagellated strain.

<p>TEM was performed using culture samples from flagellated strain χ3339 containing R995 (<b><u>Panel A</u></b>) or R995 + SPI-1 (<b><u>Panel B and C</u></b>) (size bar = 100 nm). White arrows indicate type III secretion needles among the flagella. <b><u>Panel D</u></b>: Culture tubes containing above strains and χ3339 R995 + SPI-1 <i>invA</i> strain. Note biofilm in R995 + SPI-1- containing strain. <b><u>Panel E</u></b>: Culture tubes from the experiment in Panel D were stained with crystal violet and quantitated for staining using A₅₇₀ values as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033080#s4" target="_blank">Materials and Methods</a>. A ratio of the A₅₇₀ value for each sample to the A₅₇₀ value for the R995 sample was calculated and plotted. The statistical difference between the R995 + SPI-1 and R995 + SPI-1 <i>invA</i> samples is p = 0.0003.</p

Strains and plasmids used in this study.

<p>Strains and plasmids used in this study.</p

Immunofluorescence microscopy of biofilm samples.

<p><b><u>Panel A:</u></b> Biofilms were harvested from strain χ3339 <i>flhCD</i> containing either R995 + SPI-1 SipA-FLAG or R995 + SPI-1 and visualized via immunofluorescence microscopy as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033080#s2" target="_blank">Results</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033080#s4" target="_blank">Materials and Methods</a>. DAPI (corresponding to cells), DyLight549 (corresponding to SipA-FLAG protein), or merged images are indicated. A cell clump from the harvested biofilm for each sample is indicated by the white arrow. Quantitation of red (DyLight549-stained) clumps for each sample is provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033080#pone.0033080.s002" target="_blank">Figure S2</a>. Size bar = 3000 nm. <b><u>Panel B:</u></b> Higher magnification images of biofilm cell clumps obtained from strains as indicated in Panel A. Size bar = 2000 nm.</p

R995 + SPI-1 strains express elevated SPI-1 functions.

<p><b><u>Panel A.</u></b> TEM micrographs of χ3339 <i>flhCD</i> strain containing R995 (denoted as “R”), R995 + SPI-1, or R995 + SPI-1 <i>invA</i>. Arrows indicate type III system needles on the surface of the R995 + SPI-1 strain. Size bar = 100 nm. <b><u>Panel B</u></b>. Comparison of invasion of indicated strains (χ3339 <i>flhCD</i> background) into Int407 intestinal epithelial cells. The ratio of percent invasion for each strain to the percent invasion for the R995 strain was calculated and plotted. Note that the <i>flhCD</i> mutation in this strain background renders these bacteria invasion defective since FlhCD plays a role in regulation of SPI-1 gene expression. Thus, an increase in invasion (indicating an elevation in SPI-1 activity expressed from R995-encoded SPI-1) is able to be easily observed in this strain background.</p

Biofilm-like cell aggregation mediated by SPI-1.

<p><b><u>Panel A</u>.</b> Cultures of the <i>S.</i> Typhimurium strain χ3339 <i>flhCD</i> containing either R995 (denoted as “R”), R995 + SPI-1, or R995 + SPI-1 <i>invA</i> are shown. Note adhered biofilm for the R995 + SPI-1 strain. <b><u>Panel B.</u></b> Crystal violet stains of cultures of the same strains as in panel A. After removal of non-adherent cells, the adherent biofilm cells were stained with crystal violet as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033080#s4" target="_blank">Materials and Methods</a>. <b><u>Panel C.</u></b> Cell clumps present in R995 + SPI-1 cultures (compared to R995 and R995 + SPI-1 <i>invA</i> cultures) are noted by the white arrow. <b><u>Panel D.</u></b> Quantification of crystal violet staining. After crystal violet staining of adhered cells and extraction of stain via acetic acid wash, the A₅₇₀ values of extracted stains were obtained from the indicated samples. A ratio of A₅₇₀ for each sample to the A₅₇₀ for the R995 strain was calculated and plotted. The statistical difference between the R995 + SPI-1 and R995 + SPI-1 <i>invA</i> samples is p = 0.0004. <b><u>Panel E.</u></b> Strain χ3339 <i>flhCD</i> expressing GFP (via plasmid pGreenTIR) containing R995 or R995 + SPI-1 was grown in glass tubes, and then adhered cells were visualized after removal of the broth culture using fluorescence microscopy at the same magnification (250×). Blurriness on the edges is due to the concave nature of the culture tube. Note the presence of single cells (indicated as punctate green spots) or clumps of cells on the surface of the glass that are non-adherent in the R995, GFP sample. By contrast, in the R995 + SPI-1, GFP sample, there are a massive number of cells organized into an extensive, adhered matt on the glass surface.</p

Immunogold electron microscopy and SEM analysis.

<p>Biofilm samples of strain χ3339 <i>flhCD</i> containing either R995 + SPI-1 (<b><u>Panels A and D</u></b>) or R995 + SPI-1 SipA-FLAG (<b><u>Panels B, C, and E</u></b>) were processed for immunogold electron microscopy as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033080#s4" target="_blank">Materials and Methods</a>. Panels B and C are different images obtained from the same R995 + SPI-1 SipA-FLAG sample. Size bars: Panels A, B, C = 100 nm, and Panels D, E = 500 nm. Asterisks in Panels D and E indicate bacteria. Quantification of the immunogold particles for each sample is presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033080#pone.0033080.s004" target="_blank">Figure S4</a>. Higher magnification images of immunogold particles from Panels B and E are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033080#pone.0033080.s003" target="_blank">Figure S3</a>. <b><u>Panel F</u></b> is an SEM image of the R995 + SPI-1 strain biofilm sample (size bar = 2000 nm). Arrows indicate extracellular, amorphous biofilm material.</p